么么么么

本人做大鼠肝脏SABC免疫组化，买的santacruze的一抗，博士德的二抗试剂盒和DAB。高压修复，过夜或不过夜，做了几十张片子了，大部分用1：100做，其中就1张效果还可以，背景较浅，虽然阳性细胞棕色不怎么深（但是整张片子就一处血管旁有十几个细胞阳性），但是对比还是可以的。其中几张片子就是很深的背景，但是也有棕色更深的阳性细胞，不知道那些是不是真的阳性细胞；大部分片子都是深深的背景，看不到阳性。这几天用PBS代替一抗做对照，用中衫的DAB，显色速度很快很快，并且发现阴性对照的结果和阳性片子差不多，每个细胞胞浆都有着色，血管周围细胞颜色更深一点。阴性对照如果不加二抗最后显色为蓝色。崩溃掉了。我想既然曾经做出来效果还可以的片子，一抗浓度应该就在1：100左右。会不会是内源性生物素的关系？？这几天用PBS代替一抗后用20-30%鸡蛋清封闭内源性生物素，然后再加SABC试剂盒染色，结果还是一样，每个细胞浆都有染色，血管周围细胞更深。 是不是鸡蛋清封闭后还得用牛奶？？ ***跪求原因分析。。。快2个月了，结果还是出不来，崩溃掉了。。麻烦有爱心的专业人士指点下，感激不尽。。

[ 本帖最后由 么么么么 于 2010-2-23 22:25 编辑 ]

shanghainese

Why should I block endogenous biotin
Some cells or tissues contain endogenous biotin. Using avidin-biotin method may result in high, non-specific background staining. This non-specific background can be significantly reduced by pre-treatment of cells/tissues with avidin/biotin blocking reagents prior to the incubation of biotinylated antibody

How do I know if my tissues contain endogenous biotin
Kidney, liver and spleen usually contain high level of endogenous biotin and a blocking step is needed when you are using avidin-biotin system for these tissues. You can also do a ** test by incubating sections directly with ABC complex or streptavidin-HRP and then DAB, but be sure to apply hydrogen peroxide first to rule out the background staining caused by endogenous peroxidase.

What chemicals/reagents to use for blocking endogenous biotin
Two chemicals, avidin and biotin, are needed. You can purchase from Sigma and make 0.05% avidin and 0.005% biotin in PBS. Commercial avidin/biotin blocking kits are also available from many companies. Dr. Rodney Miller has also used egg white as a source of avidin and skim milk as a source of biotin to block endogenous biotin successfully,  but water should be used to rinse sections between steps since PBS will precipitate out proteins in the egg white.

Where should I block endogenous biotin during IHC procedure
The blocking step should occur immediately after normal serum blocking and before primary antibody incubation since (1) antigen retrieval procedure may reveal some endogenous biotin; (2) normal serum can contain endogenous biotin.

What is the procedure for blocking endogenous biotin
Incubate sections in avidin solution for 15 minutes followed by brief rinse in PBS, and then incubate sections in biotin solution for 15 minutes (all at room temperature). Briefly rinse in PBS and continue the protocol with primary antibody.

Why do I need two steps to block endogenous biotin
The first step is the incubation with avidin solution and this will allow avidin to bind any avidin binding sites on biotin in the tissue, and the second step is the incubation with biotin and this step is to saturate all the biotin-binding sites left open on the avidin.

I blocked with avidin/biotin blocking reagents, but still getting background staining
The avidin/biotin solution may be expired. Try fresh solution and it may solve the problem. If the background problem persists, you may consider switching to a different detection system.

么么么么

非常谢谢版主。。感动。我明天就去试试看。。
还有两个问题：
1、前面说  but water should be used to rinse sections between steps since PBS will precipitate out proteins in the egg white.
后面Incubate sections in avidin solution for 15 minutes followed by brief rinse in PBS, and then incubate sections in biotin solution for 15 minutes (all at room temperature). Briefly rinse in PBS and continue the protocol with primary antibody.
到底是用蒸馏水冲洗呢  还是用PBS。。 还有鸡蛋清  和脱脂奶粉用PBS配好呢还是蒸馏水配制???   

2、我们平时的一般程序不是血清封闭后不能冲洗甩掉后直接加一抗的吗 ， 文章的意思好像是血清封闭后再封闭生物素再加一抗，这样会不会有影响？

多谢多谢。。。

[ 本帖最后由 么么么么 于 2010-2-14 13:36 编辑 ]

shanghainese

What chemicals/reagents to use for blocking endogenous biotin
Two chemicals, avidin and biotin, are needed. You can purchase from Sigma and make 0.05% avidin and 0.005% biotin in PBS. Commercial avidin/biotin blocking kits are also available from many companies. Dr. Rodney Miller has also used egg white as a source of avidin and skim milk as a source of biotin to block endogenous biotin successfully,  but water should be used to rinse sections between steps since PBS will precipitate out proteins in the egg white.
这一段里说的是用蛋青和去脂奶粉来阻断/封闭内源性生物素.在用蛋青和去脂奶粉这两步骤之间要用蒸馏水洗.

[ 本帖最后由 shanghainese 于 2010-2-14 13:48 编辑 ]

shanghainese

What is the procedure for blocking endogenous biotin
Incubate sections in avidin solution for 15 minutes followed by brief rinse in PBS, and then incubate sections in biotin solution for 15 minutes (all at room temperature). Briefly rinse in PBS and continue the protocol with primary antibody.
后面这一段里说的是用商品化的抗生物素avidin 和生物素 biotin 一起来来阻断/封闭内源性生物素.
这两步骤之间可以用PBS洗.

么么么么

版主老师好！版主老师新年好！
我又做了下阴性对照， 现在3%双氧水.甲醇灭活过氧化物酶，高压修复，在血清封闭后  鸡蛋清封闭25min，蒸馏水冲洗，5%脱脂奶粉封闭20min，PBS冲洗，再加PBS（代替一抗），最后结果还是一样啊，很黄。

前面说PBS会使鸡蛋清出现蛋白沉淀，我就用100ml蒸馏水配制约30ml鸡蛋清，发现出现明显的白色絮状物，好像鸡蛋清用温水泡过的感觉，我就用纱布过滤了下。后来测了蒸馏水的PH值，偏酸的，6.0左右。我想问下鸡蛋清是不是得用PH7.0的蒸馏水配制？

我买的是santacruze的一抗，网站建议用SABC染色。如果结果还是这样，是否可以换用其他的非亲和素的染色方法。

shanghainese

推荐用
http://www.vectorlabs.com/Protocols/SP2001.pdf

么么么么

版主老师啊。这个好贵。。
现在做的片子背景都是黄黄的，视野中有些细胞染色相对更深。。  阴性对照结果也还是这样。。你说 这种情况有没有可能和二抗失效有关系？？                     我一抗是santacruze小鼠抗大鼠 的，二抗是博士德羊抗小鼠的。。     我做的组织是大鼠， 据说选择小鼠一抗这样容易发生交叉反应。。唉，可惜我的指标只有santacruze能做组化。

还有个问题，santa推荐ABC染色，我能否改用福建迈新的即用型快速免疫组化MaxVisionTM检测试剂盒  或者 中杉的小鼠超敏二步法免疫组化检测试剂（与大鼠无交叉反应）PV-9005 ？？？

[ 本帖最后由 么么么么 于 2010-2-23 16:29 编辑 ]

么么么么

老师，我用小鼠的一抗做大鼠组织是不是原则性错误了？？   我的片子都是一片黄，阴性对照和阳性片结果一样这种情况的原因是不是可能就是这个？

我搜到GBI公司的超灵敏多聚HRP/AP免疫组化检测试剂盒http://www.gbichina.com/products/newproducts/immohistochemical.htm   上面说针对这类问题。。您看可行吗？

[ 本帖最后由 么么么么 于 2010-2-23 22:17 编辑 ]

shanghainese

小鼠的一抗不一定与大鼠有交叉反应。